Eukaryotic DNA transposons can be classified into ten or so superfamilies (Kapitonov & Jurka, 2004). One of the most widely distributed is the hAT transposon superfamily, which has active members in plants and insects. We began our structural studies of eukaryotic DNA transposases with Hermes, a DNA transposon that is active in not only the house fly from which it was isolated but also in other insects such as Aedes aegypti (Sarkar et al., 1997), the mosquito species that transmits yellow fever. A close relative of Hermes, the Herves transposon, is active in the malaria vector Anopheles gambiae (Arensburger et al., 2005). An active insect transposon is particularly interesting because it offers the potential to produce transgenic insects for controlling medically significant pests.[unreadable] Hermes transposition has been recapitulated in vitro and proceeds through a mechanism in which excision is accompanied by hairpin formation on the DNA flanking the transposon (Zhou et al., 2004), as also seen for the RAG1/2 recombinase of the adaptive immune system. We recently solved the structure of an N-terminally truncated version of the 612-residue Hermes protein. The protein fold revealed a four-domain protein organized around the retroviral integrase-like catalytic core characteristic of DDE transposases. The DDE catalytic core is disrupted by a large insertion domain whose structure does not resemble any other protein characterized to date, and its presence conforms to the trend that DDE transposases capable of forming hairpins on their DNA substrates require an ancillary domain to provide the amino acids needed to promote hairpin formation and to stabilize them. On the other hand, one of the paradigms seen in prokaryotic transposases - that they assemble into an active multimeric complex upon DNA binding - is not followed. Curiously, Hermes is pre-assembled as a hexamer. As we crystallized only a proteolyzed dimeric form of Hermes, we used our structural results to propose a model for the structure of the hexamer; this model is based on a ring of subunits in which neighboring pairs of subunits around the ring represent the catalytically active unit.[unreadable] Our current focus is the full-length protein and its complexes with DNA. We have recently been able to work out conditions to express and purify full-length Hermes with yields suitable for crystallization studies. Complexes of Hermes with transposon end sequences are monodisperse and highly soluble, and crystallization trials are currently underway. We have initiated studies on related hAT transposases from Aedes aegypti and Tribolium castaneum which may have biophysical properties more amenable to co-crystallization with DNA. [unreadable] We are also interested in DNA transposition systems in current use in the study of mammalian cells, including the resurrected Sleeping Beauty transposon (Ivics et al., 1997) and piggyBac (Wu et al., 2006). As the applications of these transposons can be limited by the ability to transpose in only certain cell types and non-specific targeting, we believe that X-ray structures of eukaryotic transposases can provide important insights into aspects of their mechanisms and regulation.[unreadable] [unreadable] Dupuy, A.J., Akagi, K., Largaespada, D.A., Copeland, N.G., and Jenkins, N.A. (2005) Nature 436, 221-226.[unreadable] Ivics, Z., Hackett, P.B., Plasterk, R.H., and Izsvak, Z. (1997) Cell 91, 501-510.[unreadable] Wu, S.C., Meir, Y.J., Coates, C.J., Handler, A.M., Pelczar, P., Moisyadi, S., and Kaminski, J.M. (2006) Proc. Natl. Acad. Sci. USA 103, 15008-15013.[unreadable] Kapitonov, V.V. and Jurka, J. (2004) DNA Cell Biol. 23, 311-324.[unreadable] Sarkar, A., Yardley, K., Atkinson, P.W., James, A.A., and O'Brochta, D.A. (1997) Insect Biochem. Mol. Biol. 27, 359-363.[unreadable] Zhou L.Q., Mitra R., Atkinson P.W., Hickman A.B., Dyda F., and Craig N.L. (2004) Nature 432, 995-1001.